Primary Goal The goal of this project is to unravel the nucleocytoplasmic transport mechanism and to advance our understanding of how transport maintains or through dysfunction fails to maintain in cells. The advanced knowledge obtained by these investigations provides the foundation for further disease diagnosis, treatment and prevention. Intellectual Merit Nuclear pore complexes (NPCs) span the nuclear envelope and enable bidirectional trafficking between the cytoplasm and nucleus in eukaryotic cells. Small molecules (< 20-40 kDa) passively diffuse through the NPCs. Translocation of larger molecules (up to 50 MDa) is hindered by the phenylalanine- glycine (FG) nucleoporin (Nups) barrier inside the NPCs unless they are chaperoned by transport receptors. However, challenged by measuring the spatial structure of FG repeats and a series of transient interactions between the transport receptors and the FG repeats, the precise transport mechanism remains in dispute as to how different-sized molecules pave their pathways through the permeable barrier of the NPC. To unravel the transport mechanism, principal Investigator develops an innovative single molecule method to meet the major challenges. Research Approach The principal Investigator's lab develops a novel single-molecule approach, single-point edge-excitation sub-diffraction (SPEED) microscopy, to test nuclear transport models with a spatiotemporal resolution of 9 nm and 400 ms. SPEED microscopy enables us to capture transient interactions occurred in the NPC under real- time trafficking conditions that have escaped detection by previous single-molecule methods or electron microscopy. Principal Investigator proposes studies with the following SPECIFIC AIMS: (1) To determine the 3D spatial locations of transport pathways for small molecules in permeabilized cells. (2) To determine the 3D spatial locations of transport pathways for more transport receptors and their cargo complexes in permeabilized cells. (3) To characterize the 3D conformational change of FG-Nups at various transport receptor concentrations in permeabilized cells. (4) To determine the 3D pathways for small molecules, transport receptors and facilitated translocations in live cells. Specific aim 1, 2 & 4 will examine the hypothesis that small molecules diffuse through a central channel, and that small and large molecules spatially separate the pathways. Specific aim 3 & 4 will specifically test whether the transport receptors collapse the FG-repeats filaments or dissolve into filamentous meshwork. Broader Impacts Dysfunctions of the nuclear transport system are linked to numerous human diseases including leukemias, cancers, and primary biliary cirrhosis. Success of this project will significantly change the landscape of research on nuclear transport and also provide new approaches to biomedical imaging, drug development, and medical diagnostics and treatments of these human diseases.